There are over 35 mouse homeobox-containing genes known as Hox. The Hox genes are divided into four multigene dusters (Hox-A, -B, -C, & -D), which reside on four different chromosomes. These genes are expressed in restricted patterns during embryogenesis. The function of Hox genes during mammalian development was first studied by overexpressing the Hoxa-4 gene in transgenic mice (Wolgemuth et al., 1989). That study demonstrated for the first time that the expression of a mammalian homeobox-containing gene could alter development. Since then, studies using gain of function and loss of function transgenic mice have suggested that the Hox genes constitute a complex genetic network for patterning the mouse embryo. In this application, experiments are proposed to study the individual and combined roles of a specific class of Hox genes during mouse development. This class of mouse Hox genes is known as the Deformed class or Group 4, which includes the four Hox genes Hoxa-4, b-4, c-4, and d-4. These Hox genes possess a homeodomain most similar to Drosophila Deformed and constitute a paralogous group of genes between the clusters. Gene targeting experiments have been initiated using mouse embryonic stem (ES) cells to generate Hoxa-4, c-4, and d-4 mutations in the germ line of mice to study their required functions during mammalian development. Germ line chimeras derived from Hoxa-4 mutant ES cells have already been generated. In addition to the individual Hox phenotypes, mice carrying these three Hox mutations and the recently reported Hoxb-4 mutation (Ramirez-Solis et al., 1993) will be interbred to produce mice deficient for two or more of these genes. The production of mouse strains that possess the four Deformed-class Hox gene mutations will provide a powerful resource for studying the function of Hox genes. These focused studies should provide important information about the roles of a specific class of Hox genes during mouse development. The information gained in this model system should be useful in understanding normal and abnormal human development.